Steric regulation of CRISPR/Cas12a trans-cleavage kinetics via split-activator extensions.

ABSTRACT: Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a holds substantial promise for molecular diagnostics, yet its rapid and uncontrolled activation often results in background leakage and disrupts the coordination of upstream reaction modules. Here, we established a steric-regulation framework that enables predictable tuning of Cas12a trans-cleavage kinetics through rationally engineered extensions on split activators. Systematic analysis of extension orientation, length, and hybridization state revealed quantitative and direction-dependent rules governing steric control of activator assembly and Cas12a activation. Guided by these insights, we integrated the sterically regulated split activator into an entropy-driven DNA circuit to construct a fully one-pot cascaded de

SUBMITTER: Zhang J 

PROVIDER: S-EPMC12802953 | biostudies-literature | 2026 Jan

REPOSITORIES: biostudies-literature